Haipeng Cao

Bdellovibrios, potential biocontrol bacteria against pathogenic Aeromonas hydrophila

Recent studies have revealed that the use of bdellovibrios is an alternative to control bacteriosis. However, no bdellovibrios are available against Aeromonas hydrophila infections in sturgeons. In the present study, a potential Bdellovibrio strain F16 was isolated from sturgeon gut samples, using a sturgeon-pathogenic A. hydrophila as the prey bacterium. It was initially identified as a Bdellovibrio strain using morphological characteristics and specific PCR amplification, and confirmed to be Bdellovibrio sp. strain ETB (GenBank Accession No. DQ302728) and Bdellovibrio bacteriovorus strain SRA9 (GenBank Accession No. AF263833) by phylogenetic analysis. In addition, it was shown to be safe for mammalians and sturgeons, had a wide prey range, and exhibited significant bacteriolytic effects on the pathogenic A. hydrophila. To the best of our knowledge, this is the first report on a promising gut Bdellovibrio strain against pathogenic A. hydrophila.

Bacillus amyloliquefaciens G1: A Potential Antagonistic Bacterium against Eel-Pathogenic Aeromonas hydrophila

Recent studies have revealed that the use of probiotics is an alternative to control marine aeromonas. However, few probiotics are available against Aeromonas hydrophila infections in eels. In the present study, a potential antagonistic strain G1 against the eel-pathogenic A. hydrophila was isolated from sediment underlying brackish water. Its extracellular products with antibacterial activities were shown to be stable under wide range of pH, temperature, and proteinase K. It was initially identified as Bacillus amyloliquefaciens using API identification kits and confirmed to be B. amyloliquefaciens strain (GenBank accession number DQ422953) by phylogenetic analysis. In addition, it was shown to be safe for mammalians, had a wide anti-A. hydrophila spectrum, and exhibited significant effects on inhibiting the growth of the eel-pathogenic A. hydrophila both in vitro and in vivo. To the best of our knowledge, this is the first report on a promising antagonistic Bacillus amyloliquefaciens strain from brackish water sediment against eel-pathogenic A. hydrophila.

Grass carp (Ctenopharyngodon idellus) infected with multiple strains of Aeromonas hydrophila

Ctenopharyngodon idellus is an important commercial fish species in China and many other countries to meet human food habits. However, there is no definitive data to indicate the multi-infection of pathogenic Aeromonas hydrophila strains in cultured C. idellus. In this study, four strong virulent strains with five virulence genes were simultaneously isolated from the cultured C. idellus suffering from septicemia, and identified as different A. hydrophila isolates using the ATB 32GN system, phylogenetic analysis, enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). They were susceptible to chloramphenicol, tobramycin, kanamycin, norfloxacin, spectinomycin, furantoin, azithromycin, and resistant to carbenicillin, penbritin, clindamycin. In addition, to further control the multi-infection of A. hydrophila, norfloxacin, the well-known fishery drug widespread used in aquaculture, was employed to treat this disease. Its minimum inhibitory concentrations (MICs) were 2~4 mg/L and good protection effects were also exhibited on the naturally infected fish at a final concentration of 1 and 2 g/Kg in the feed (cal. 20 and 40 mg/Kg of fish). As an important pathogen of a zoonotic and foodborne disease, the multi-infection of A. hydrophila strains may be an emerging threat in grass carp farming and food safety, and more importance should be attached to their multi-infection in fish farming.

Vibrio cholerae pathogen from the freshwater-cultured whiteleg shrimp Penaeus vannamei and control with Bdellovibrio bacteriovorus.

Vibriosis has become a major global economic problem in freshwater-farmed whiteleg shrimp (Penaeus vannamei). The prevention and control of vibriosis are now priority research topics. In this study, a pathogenic strain (QH) was isolated from vibriosis-infected freshwater-farmed P. vannamei that resulted in leg yellowing and was identified as a Vibrio cholerae isolate through phylogenetic analysis and the API 32GN system. A phylogenetic tree that was constructed using the neighbor-joining method further confirmed the QH isolate as a V. cholerae strain. A virulent outer membrane protein (ompU) gene was found to be present in the QH isolate, which further confirmed its pathogenicity. In addition, Bdellovibrio bacteriovorus conferred significant protection against V. cholerae: B. bacteriovorus exhibited significant bacteriolytic effects on the V. cholerae pathogen, possessed a wide prey range that included Vibrio pathogens, and displayed a positive protective efficacy against experimental V. cholerae infection in P. vannamei. To the best of our knowledge, this is the first report of the control of shrimp pathogen V. cholerae with B. bacteriovorus.

Identification of a Proteus penneri isolate as the causal agent of red body disease of the cultured white shrimp Penaeus vannamei and its control with Bdellovibrio bacteriovorus

Bacteriosis has become a major economic problem in the farming of the Pacific white shrimp Penaeus vannamei. However, no definitive data are available about Proteus penneri infection in cultured P. vannamei and its control. In this study, a virulent strain NC was isolated from diseased P. vannamei suffering from red body disease and identified as a P. penneri isolate through phylogenetic analysis and ATB 32GN system. A phylogenetic constructed tree using the neighbour-joining method identified the NC isolate as a P. penneri strain. In addition, Bdellovibrio bacteriovorus conferred significant protection against P. penneri: it exhibited significant bacteriolytic effects on the pathogenic P. penneri, had a wide prey range towards Proteus pathogens, and displayed a good protective efficacy on experimental P. penneri infection in P. vannamei. To the best of our knowledge, this is the first report of farmed P. vannamei infected with P. penneri and its control with B. bacteriovorus.

Identification of up-regulated proteins potentially involved in the antagonism mechanism of Bacillus amyloliquefaciens G1

The use of Bacillus probiotics has been demonstrated as a promising method in the biocontrol of bacterial diseases in aquaculture. However, the molecular antibacterial mechanism of Bacillus still remains unclear. In order to explore the antibacterial mechanism of the potential antagonistic Bacillus amyloliquefaciens strain G1, comparative proteomics between B. amyloliquefaciens strain G1 and its non-antagonistic mutant strain was investigated. The 2-dimensional electrophoresis gel maps of their total extracted proteins were described and 42 different proteins were found to be highly expressed in strain G1 in comparison with those in the mutant strain. 35 of these up-regulated proteins were successfully identified using MALDI-TOF-TOF MS and databank analysis, and their biological functions were analyzed through the KEGG database. The increased expression of these proteins suggested that high levels of energy metabolism, biosynthesis and stress resistance could play important roles in strain G1’s antagonism. To our knowledge, this is the first report on the proteins involved in the antagonism mechanism of B. amyloliquefaciens using a proteomic approach and the proteomic data also contribute to a better understanding of the molecular basis for the antagonism of B. amyloliquefaciens.

Saprolegnia australis R. F. Elliott 1968 infection in Prussian carp Carassius gibelio (Bloch, 1782) eggs and its control with herb extracts

Summary In order to control saprolegniosis in Prussian carp (Carassius gibelio (Bloch, 1782) eggs, it is important to screen herb extracts as potential anti‐Saprolegnia drugs in Prussian carp hatcheries. For this purpose, an oomycete water mould (strain SC) isolated from Prussian carp [Carassius gibelio (Bloch, 1782)] eggs suffering from saprolegniosis was characterised morphologically as well as from ITS rDNA sequence data. Initially identified as a Saprolegnia sp. based on its morphological features, the constructed phylogenetic tree using the neighbour joining method further indicated that the SC strain was closely related to Saprolegnia australis R. F. Elliott 1968 strain VI05733 (GenBank accession no. HE798564), and which could form biofilm communities as virulence factors. In addition, aqueous extracts from forty Chinese herbs were screened as possible anti‐Saprolegnia agents. Among them, a 1 g ml−1 extract from Radix sanguisorbae was the most efficacious anti‐Saprolegnia agent, indicated by the minimum inhibitory concentration that was as low as 256 mg L−1. Relative survival of 73 and 88% was obtained against the SC strain in fish eggs at concentrations of 256 and 1280 mg L−1, respectively. This is the first known report of Saprolegnia australis R. F. Elliott 1968 infection in C. gibelio (Bloch, 1782) eggs involving the screening of R. sanguisorbae extracts as potential anti‐Saprolegnia agents.

Effect of copper sulfate on Bdellovibrio growth and bacteriolytic activity towards gibel carp-pathogenic Aeromonas hydrophila

The use of bdellovibrios has been regarded as an alternative to control multidrug-resistant pathogens and fish bacteriosis. However, scarce information is available on the potential of bdellovibrios in the presence of copper sulfate, which is an algicide widely used to treat cyanobacterial blooms in aquaculture. In the present study, the effects of copper sulfate at sublethal and lethal levels (0.1 and 1.0 mg·L-1) on Bdellovibrio sp. strain BDF-H16 were evaluated. The growth of Bdellovibrio sp. strain BDF-H16 was significantly promoted by both concentrations of copper sulfate, but less so by the lethal concentration. The bacteriolysis of gibel carp-pathogenic Aeromonas hydrophila by Bdellovibrio sp. strain BDF-H16 was also stimulated by copper sulfate in both solid and liquid media. However, Bdellovibrio sp. strain BDF-H16 with 0.1 mg·L-1 copper sulfate clearly inhibited infection of gibel carps by A. hydrophila better than Bdellovibrio sp. strain BDF-H16 with 1.0 mg·L-1 copper sulfate did.